COMBINATIONS INCLUDING Cry34Ab/35Ab AND Cry6Aa PROTEINS TO PREVENT DEVELOPMENT OF RESISTANCE IN CORN ROOTWORMS (DIABROTICA SPP.)

ABSTRACT

The subject invention relates in part to Cry34Ab/35Ab in combination with Cry6Aa. The subject invention relates in part to the surprising discovery that combinations of Cry34Ab/Cry35Ab and Cry6Aa are useful for preventing development of resistance (to either insecticidal protein system alone) by a corn rootworm ( Diabrotica  spp.) population. Included within the subject invention are plants producing these insecticidal Cry proteins, which are useful to mitigate concern that a corn rootworm population could develop that would be resistant to either of these insecticidal protein systems alone. Plants (and acreage planted with such plants) that produce these two insecticidal protein systems are included within the scope of the subject invention. The subject invention also relates in part to combinations of Cry34Ab/35Ab and Cry3Aa proteins “triple stacked” with a Cry6Aa protein. Transgenic plants, including corn, comprising a cry6Aa gene, cry34Ab/35Ab genes, and a cry3Aa gene are included within the scope of the subject invention. Thus, such embodiments target rootworms with three modes of action.

BACKGROUND

Humans grow corn for food and energy applications. Corn is an importantcrop. It is an important source of food, food products, and animal feedin many areas of the world. Insects eat and damage plants and therebyundermine these human efforts. Billions of dollars are spent each yearto control insect pests and additional billions are lost to the damagethey inflict.

Damage caused by insect pests is a major factor in the loss of theworld's corn crops, despite the use of protective measures such aschemical pesticides. In view of this, insect resistance has beengenetically engineered into crops such as corn in order to controlinsect damage and to reduce the need for traditional chemicalpesticides.

Over 10 million acres of U.S. corn are infested with corn rootwormspecies complex each year. The corn rootworm species complex includesthe northern corn rootworm (Diabrotica barberi), the southern cornrootworm (D. undecimpunctata howardi), and the western corn rootworm (D.virgifera virgifera). (Other species include Diabrotica virgifera zeae(Mexican corn rootworm), Diabrotica balteata (Brazilian corn rootworm),and Brazilian corn rootworm complex (Diabrotica viridula and Diabroticaspeciosa).)

The soil-dwelling larvae of these Diabrotica species feed on the root ofthe corn plant, causing lodging. Lodging eventually reduces corn yieldand often results in death of the plant. By feeding on cornsilks, theadult beetles reduce pollination and, therefore, detrimentally affectthe yield of corn per plant. In addition, adults and larvae of the genusDiabrotica attack cucurbit crops (cucumbers, melons, squash, etc.) andmany vegetable and field crops in commercial production as well as thosebeing grown in home gardens.

Synthetic organic chemical insecticides have been the primary tools usedto control insect pests but biological insecticides, such as theinsecticidal proteins derived from Bacillus thuringiensis (Bt), haveplayed an important role in some areas. The ability to produceinsect-resistant plants through transformation with Bt insecticidalprotein genes has revolutionized modern agriculture and heightened theimportance and value of insecticidal proteins and their genes.

Insecticidal crystal proteins from some strains of Bacillusthuringiensis (B.t.) are well-known in the art. See, e.g., Hofte et al.,Microbial Reviews, Vol. 53, No. 2, pp. 242-255 (1989). These proteinsare typically produced by the bacteria as approximately 130 kDaprotoxins that are then cleaved by proteases in the insect midgut, afteringestion by the insect, to yield a roughly 60 kDa core toxin. Theseproteins are known as crystal proteins because distinct crystallineinclusions can be observed with spores in some strains of B.t. Thesecrystalline inclusions are often composed of several distinct proteins.

One group of genes which have been utilized for the production oftransgenic insect resistant crops are the delta-endotoxins from Bacillusthuringiensis (B.t.). Delta-endotoxins have been successfully expressedin crop plants such as cotton, potatoes, rice, sunflower, as well ascorn, and have proven to provide excellent control over insect pests.(Perlak, F. J et al. (1990) Bio/Technology 8, 939-943; Perlak, F. J. etal. (1993) Plant Mol. Biol. 22: 313-321; Fujimoto H. et al. (1993)Bio/Technology 11: 1151-1155; Tu et al. (2000) Nature Biotechnology18:1101-1104; PCT publication number WO 01/13731; and Bing J W et al.(2000) Efficacy of Cry1F Transgenic Maize, 14^(th) BiennialInternational Plant Resistance to Insects Workshop, Fort Collins, Colo.)

Several Bt proteins have been used to create the insect-resistanttransgenic plants that have been successfully registered andcommercialized to date. These include Cry1Ab, Cry1Ac, Cry1F, Cry1A.105,Cry2Ab, Cry3Aa, Cry3Bb, and Cry34/35Ab in corn, Cry1Ac and Cry2Ab incotton, and Cry3A in potato.

The commercial products expressing these proteins express a singleprotein except in cases where the combined insecticidal spectrum of 2proteins is desired (e.g., Cry1Ab and Cry3Bb in corn combined to provideresistance to lepidopteran pests and rootworm, respectively) or wherethe independent action of the proteins makes them useful as a tool fordelaying the development of resistance in susceptible insect populations(e.g., Cry1Ac and Cry2Ab in cotton combined to provide resistancemanagement for tobacco budworm).

Some of the qualities of insect-resistant transgenic plants that haveled to rapid and widespread adoption of this technology also give riseto the concern that pest populations will develop resistance to theinsecticidal proteins produced by these plants. Several strategies havebeen suggested for preserving the utility of Bt-based insect resistancetraits which include deploying proteins at a high dose in combinationwith a refuge, and alternation with, or co-deployment of, differenttoxins (McGaughey et al. (1998), “B.t. Resistance Management,” NatureBiotechnol. 16:144-146).

The proteins selected for use in an Insect Resistance Management (IRM)stack should be active such that resistance developed to one proteindoes not confer resistance to the second protein (i.e., there is notcross resistance to the proteins). If, for example, a pest populationselected for resistance to “Protein A” is sensitive to “Protein B”, onewould conclude that there is not cross resistance and that a combinationof Protein A and Protein B would be effective in delaying resistance toProtein A alone.

In the absence of resistant insect populations, assessments can be madebased on other characteristics presumed to be related tocross-resistance potential. The utility of receptor-mediated binding inidentifying insecticidal proteins likely to not exhibit cross resistancehas been suggested (van Mellaert et al. 1999). The key predictor of lackof cross resistance inherent in this approach is that the insecticidalproteins do not compete for receptors in a sensitive insect species.

In the event that two Bt toxins compete for the same receptor, then ifthat receptor mutates in that insect so that one of the toxins no longerbinds to that receptor and thus is no longer insecticidal against theinsect, it might be the case that the insect will also be resistant tothe second toxin (which competitively bound to the same receptor). Thatis, the insect is said to be cross-resistant to both Bt toxins. However,if two toxins bind to two different receptors, this could be anindication that the insect would not be simultaneously resistant tothose two toxins.

A relatively newer insecticidal protein system was discovered inBacillus thuringiensis as disclosed in WO 97/40162. This systemcomprises two proteins—one of approximately 14-15 kDa and the other ofabout 44-45 kDa. See also U.S. Pat. Nos. 6,083,499 and 6,127,180. Theseproteins have now been assigned to their own classes, and accordinglyreceived the Cry designations of Cry34 and Cry35, respectively. SeeCrickmore et al. website (biols.susx.ac.uk/home/Neil_Crickmore/Bt/).Many other related proteins of this type of system have now beendisclosed. See e.g. U.S. Pat. No. 6,372,480; WO 01/14417; and WO00/66742. Plant-optimized genes that encode such proteins, wherein thegenes are engineered to use codons for optimized expression in plants,have also been disclosed. See e.g. U.S. Pat. No. 6,218,188.

The exact mode of action of the Cry34/35 system has yet to bedetermined, but it appears to form pores in membranes of insect gutcells. See Moellenbeck et al., Nature Biotechnology, vol. 19, p. 668(July 2001); Masson et al., Biochemistry, 43 (12349-12357) (2004). Theexact mechanism of action remains unclear despite 3D atomic coordinatesand crystal structures being known for a Cry34 and a Cry35 protein. SeeU.S. Pat. Nos. 7,524,810 and 7,309,785. For example, it is unclear ifone or both of these proteins bind a typical type of receptor, such asan alkaline phosphatase or an aminopeptidase.

Furthermore, because there are different mechanisms by which an insectcan develop resistance to a Cry protein (such as by alteredglycosylation of the receptor [see Jurat-Fuentes et al. (2002) 68 AEM5711-5717], by removal of the receptor protein [see Lee et al. (1995) 61AEM 3836-3842], by mutating the receptor, or by other mechanisms [seeHeckel et al., J. Inv. Pathol. 95 (2007) 192-197]), it was impossible toa priori predict whether there would be cross-resistance betweenCry34/35 and other Cry proteins. Lefko et al. discusses a complexresistance phenomenon in rootworm. J. Appl. Entomol. 132 (2008) 189-204.

Predicting competitive binding for the Cry34/35 system is also furthercomplicated by the fact that two proteins are involved in the Cry34/35binary system. Again, it is unclear if and how these proteinseffectively bind the insect gut/gut cells, and if and how they interactwith or bind with each other.

Other options for controlling coleopterans include Cry3Bb toxins, Cry3C,Cry6B, ET29, ET33 with ET34, TIC407, TIC435, TIC417, TIC901, TIC1201,ET29 with TIC810, ET70, ET76 with ET80, TIC851, and others. RNAiapproaches have also been proposed. See e.g. Baum et al., NatureBiotechnology, vol. 25, no. 11 (November 2007) pp. 1322-1326.

Meihls et al. suggest the use of refuges for resistance management incorn rootworm. PNAS (2008) vol. 105, no. 49, 19177-19182.

BRIEF SUMMARY

The subject invention relates in part to Cry34Ab/35Ab in combinationwith Cry6Aa. The subject invention relates in part to the surprisingdiscovery that Cry34Ab/Cry35Ab and Cry6Aa are useful for preventingdevelopment of resistance (to either insecticidal protein system alone)by a corn rootworm (Diabrotica spp.) population. As one skilled in theart will recognize with the benefit of this disclosure, plants producingthese insecticidal Cry proteins will be useful to mitigate concern thata corn rootworm population could develop that would be resistant toeither of these insecticidal protein systems alone.

The subject invention is supported in part by the discovery thatcomponents of these Cry protein systems do not compete with each otherfor binding corn rootworm gut receptors.

The subject invention also relates in part to triple stacks or“pyramids” of three (or more) toxin systems, with Cry34Ab/Cry35Ab andCry6Aa being the base pair. Thus, plants (and acreage planted with suchplants) that produce these two insecticidal protein systems are includedwithin the scope of the subject invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Binding of ¹²⁵I-Cry35Ab1 (A) and ¹²⁵I-Cry6Aa1 (B) as a functionof input radio-labeled Cry toxins to BBMV prepared from western cornrootworm larvae. Specific binding=total binding−non-specific binding,error bar=SEM (standard error of mean).

FIG. 2. Binding of ¹²⁵I-Cry35Ab1 to BBMV prepared from western cornrootworm larvae at different concentrations of non-labeled competitor(log 0.1=−1.0, log 10=1.0, log 100=2.0, log 1,000=3.0).

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1: Full length, native Cry35Ab1 protein sequence

SEQ ID NO:2: Chymotrypsin-truncated Cry35Ab1 core protein sequence

SEQ ID NO:3: Full length, native Cry34Ab1 protein sequence

SEQ ID NO:4: Full length, native Cry6Aa1 protein sequence

DETAILED DESCRIPTION

Sequences for the Cry34Ab/35Ab protein are obtainable from Bacillusthuringiensis isolate PS149B1, for example. For other genes, proteinsequences, and source isolates for use according to the subjectinvention, see the Crickmore et al. website(lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/intro.html), for example.

The subject invention includes the use of Cry34Ab/35Ab insecticidalproteins in combination with a Cry6Aa toxin to protect corn from damageand yield loss caused by corn rootworm feeding by corn rootwormpopulations that might develop resistance to either of these Cry proteinsystems alone (without the other).

The subject invention thus teaches an Insect Resistance Management (IRM)stack to prevent the development of resistance by corn rootworm toCry6Aa and/or Cry34Ab/35Ab.

The present invention provides compositions for controlling rootwormpests comprising cells that produce a Cry6Aa toxin protein and aCry34Ab/35Ab toxin system.

The invention further comprises a host transformed to produce both aCry6Aa protein and a Cry34Ab/35Ab binary toxin, wherein said host is amicroorganism or a plant cell.

It is additionally intended that the invention provides a method ofcontrolling rootworm pests comprising contacting said pests or theenvironment of said pests with an effective amount of a composition thatcontains a Cry6Aa protein and further contains a Cry34Ab/35Ab binarytoxin.

An embodiment of the invention comprises a maize plant comprising aplant-expressible gene encoding a Cry34Ab/35Ab binary toxin and aplant-expressible gene encoding a Cry6Aa protein, and seed of such aplant.

A further embodiment of the invention comprises a maize plant wherein aplant-expressible gene encoding a Cry34Ab/35Ab binary toxin and aplant-expressible gene encoding a Cry6Aa protein have been introgressedinto said maize plant, and seed of such a plant.

As described in the Examples, competitive receptor binding studies usingradiolabeled Cry35Ab core toxin protein show that the Cry6Aa core toxinprotein does not compete for binding in CRW insect tissue samples towhich Cry35Ab binds. See FIG. 2. These results indicate that thecombination of Cry6Aa and Cry34Ab/35Ab proteins is an effective means tomitigate the development of resistance in CRW populations to eitherprotein system alone.

Thus, based in part on the data described above and elsewhere herein,Cry34Ab/35Ab and Cry6Aa proteins can be used to produce IRM combinationsfor prevention or mitigation of resistance development by CRW. Otherproteins can be added to this combination to expand insect-controlspectrum, for example. The subject pair/combination can also be used insome preferred “triple stacks” or “pyramid” in combination with yetanother protein for controlling rootworms, such as Cry3Ba and/or Cry3Aa.RNAi against rootworms is a still further option. See e.g. Baum et al.,Nature Biotechnology, vol. 25, no. 11 (November 2007) pp. 1322-1326.Thus, the subject combinations provide multiple modes of action againsta rootworm.

In light of the disclosure of U.S. Ser. No. 61/327,240 (filed Apr. 23,2010) relating to combinations of Cry34Ab/35Ab and Cry3Aa proteins, U.S.Ser. No. 61/476,005 (filed Apr. 15, 2011) relating to combinations ofCry34Ab/35Ab and Cry3Ba proteins, and U.S. Ser. No. 61/477,447 (filedApr. 20, 2011) relating to combinations of Cry3Aa and Cry6Aa, somepreferred “triple stacks” of the subject invention include a Cry6Aaprotein combined with Cry34Ab/35Ab and Cry3Aa and/or Cry3Ba proteins.Transgenic plants, including corn, comprising a cry6Aa gene,cry34Ab/35Ab genes, and a cry3Aa and/or cry3Ba gene are included withinthe scope of the subject invention. Thus, such embodiments target theinsect with three modes of action. Furthermore, in light of these dataand teachings, one could substitute Cry3Ba or Cry3Aa in place of theCry6Aa exemplified herein as the base combination pairing withCry34A/35A.

Deployment options of the subject invention include the use of Cry6Aaand Cry34Ab/35Ab proteins in corn-growing regions where Diabrotica spp.are problematic. Another deployment option would be to use one or bothof the Cry6Aa and Cry34Ab/35Ab proteins in combination with othertraits.

A person skilled in this art will appreciate that Bt toxins, even withina certain class such as Cry6Aa and Cry34Ab/35Ab can vary to some extent.

Genes and toxins. The term “isolated” refers to a polynucleotide in anon-naturally occurring construct, or to a protein in a purified orotherwise non-naturally occurring state. The genes and toxins usefulaccording to the subject invention include not only the full lengthsequences disclosed but also fragments of these sequences, variants,mutants, and fusion proteins which retain the characteristic pesticidalactivity of the toxins specifically exemplified herein. As used herein,the terms “variants” or “variations” of genes refer to nucleotidesequences which encode the same toxins or which encode equivalent toxinshaving pesticidal activity. As used herein, the term “equivalent toxins”refers to toxins having the same or essentially the same biologicalactivity against the target pests as the claimed toxins. The sameapplies to Cry3's if used in triple stacks according to the subjectinvention. Domains/subdomains of these proteins can be swapped to makechimeric proteins. See e.g. U.S. Pat. Nos. 7,309,785 and 7,524,810regarding Cry34/35 proteins. The '785 patent also teaches truncatedCry35 proteins. Truncated toxins are also exemplified herein.

As used herein, the boundaries represent approximately 95% (Cry6Aa's andCry34Ab's and Cry35Ab's), 78% (Cry6A's and Cry 34A's and Cry35A's), and45% (Cry6's and Cry 34's and Cry 35's) sequence identity, per “Revisionof the Nomenclature for the Bacillus thuringiensis Pesticidal CrystalProteins,” N. Crickmore, D. R. Zeigler, J. Feitelson, E. Schnepf, J. VanRie, D. Lereclus, J. Baum, and D. H. Dean. Microbiology and MolecularBiology Reviews (1998) Vol 62: 807-813. The same applies to Cry3's ifused in triple stacks, for example, according to the subject invention.

It should be apparent to a person skilled in this art that genesencoding active toxins can be identified and obtained through severalmeans. The specific genes or gene portions exemplified herein may beobtained from the isolates deposited at a culture depository. Thesegenes, or portions or variants thereof, may also be constructedsynthetically, for example, by use of a gene synthesizer. Variations ofgenes may be readily constructed using standard techniques for makingpoint mutations. Also, fragments of these genes can be made usingcommercially available exonucleases or endonucleases according tostandard procedures. For example, enzymes such as Bal31 or site-directedmutagenesis can be used to systematically cut off nucleotides from theends of these genes. Genes that encode active fragments may also beobtained using a variety of restriction enzymes. Proteases may be usedto directly obtain active fragments of these protein toxins.

Fragments and equivalents which retain the pesticidal activity of theexemplified toxins would be within the scope of the subject invention.Also, because of the redundancy of the genetic code, a variety ofdifferent DNA sequences can encode the amino acid sequences disclosedherein. It is well within the skill of a person trained in the art tocreate these alternative DNA sequences encoding the same, or essentiallythe same, toxins. These variant DNA sequences are within the scope ofthe subject invention. As used herein, reference to “essentially thesame” sequence refers to sequences which have amino acid substitutions,deletions, additions, or insertions which do not materially affectpesticidal activity. Fragments of genes encoding proteins that retainpesticidal activity are also included in this definition.

A further method for identifying the genes encoding the toxins and geneportions useful according to the subject invention is through the use ofoligonucleotide probes. These probes are detectable nucleotidesequences. These sequences may be detectable by virtue of an appropriatelabel or may be made inherently fluorescent as described inInternational Application No. WO93/16094. As is well known in the art,if the probe molecule and nucleic acid sample hybridize by forming astrong bond between the two molecules, it can be reasonably assumed thatthe probe and sample have substantial homology. Preferably,hybridization is conducted under stringent conditions by techniqueswell-known in the art, as described, for example, in Keller, G. H., M.M. Manak (1987) DNA Probes, Stockton Press, New York, N.Y., pp. 169-170.Some examples of salt concentrations and temperature combinations are asfollows (in order of increasing stringency): 2× SSPE or SSC at roomtemperature; 1× SSPE or SSC at 42° C.; 0.1× SSPE or SSC at 42° C.; 0.1×SSPE or SSC at 65° C. Detection of the probe provides a means fordetermining in a known manner whether hybridization has occurred. Such aprobe analysis provides a rapid method for identifying toxin-encodinggenes of the subject invention. The nucleotide segments which are usedas probes according to the invention can be synthesized using a DNAsynthesizer and standard procedures. These nucleotide sequences can alsobe used as PCR primers to amplify genes of the subject invention.

Variant toxins. Certain toxins of the subject invention have beenspecifically exemplified herein. Since these toxins are merely exemplaryof the toxins of the subject invention, it should be readily apparentthat the subject invention comprises variant or equivalent toxins (andnucleotide sequences coding for equivalent toxins) having the same orsimilar pesticidal activity of the exemplified toxin. Equivalent toxinswill have amino acid homology with an exemplified toxin. This amino acididentity will typically be greater than 75%, or preferably greater than85%, preferably greater than 90%, preferably greater than 95%,preferably greater than 96%, preferably greater than 97%, preferablygreater than 98%, or preferably greater than 99% in some embodiments.The amino acid identity will typically be highest in critical regions ofthe toxin which account for biological activity or are involved in thedetermination of three-dimensional configuration which ultimately isresponsible for the biological activity. In this regard, certain aminoacid substitutions are acceptable and can be expected if thesesubstitutions are in regions which are not critical to activity or areconservative amino acid substitutions which do not affect thethree-dimensional configuration of the molecule. For example, aminoacids may be placed in the following classes: non-polar, unchargedpolar, basic, and acidic. Conservative substitutions whereby an aminoacid of one class is replaced with another amino acid of the same typefall within the scope of the subject invention so long as thesubstitution does not materially alter the biological activity of thecompound. Table 1 provides a listing of examples of amino acidsbelonging to each class.

TABLE 1 Class of Amino Acid Examples of Amino Acids Nonpolar Ala, Val,Leu, Ile, Pro, Met, Phe, Trp Uncharged Polar Gly, Ser, Thr, Cys, Tyr,Asn, Gln Acidic Asp, Glu Basic Lys, Arg, His

In some instances, non-conservative substitutions can also be made. Thecritical factor is that these substitutions must not significantlydetract from the biological activity of the toxin.

Recombinant hosts. The genes encoding the toxins of the subjectinvention can be introduced into a wide variety of microbial or planthosts. Expression of the toxin gene results, directly or indirectly, inthe intracellular production and maintenance of the pesticide. Conjugaltransfer and recombinant transfer can be used to create a Bt strain thatexpresses both toxins of the subject invention. Other host organisms mayalso be transformed with one or both of the toxin genes then used toaccomplish the synergistic effect. With suitable microbial hosts, e.g.,Pseudomonas, the microbes can be applied to the situs of the pest, wherethey will proliferate and be ingested. The result is control of thepest. Alternatively, the microbe hosting the toxin gene can be treatedunder conditions that prolong the activity of the toxin and stabilizethe cell. The treated cell, which retains the toxic activity, then canbe applied to the environment of the target pest.Non-regenerable/non-totipotent plant cells from a plant of the subjectinvention (comprising at least one of the subject IRM genes) areincluded within the subject invention.

Plant transformation. A preferred embodiment of the subject invention isthe transformation of plants with genes encoding the subjectinsecticidal protein or its variants. The transformed plants areresistant to attack by an insect target pest by virtue of the presenceof controlling amounts of the subject insecticidal protein or itsvariants in the cells of the transformed plant. By incorporating geneticmaterial that encodes the insecticidal properties of the B.t.insecticidal toxins into the genome of a plant eaten by a particularinsect pest, the adult or larvae would die after consuming the foodplant. Numerous members of the monocotyledonous and dicotyledonousclassifications have been transformed. Transgenic agronomic crops aswell as fruits and vegetables are of commercial interest. Such cropsinclude, but are not limited to, maize, rice, soybeans, canola,sunflower, alfalfa, sorghum, wheat, cotton, peanuts, tomatoes, potatoes,and the like. Several techniques exist for introducing foreign geneticmaterial into plant cells, and for obtaining plants that stably maintainand express the introduced gene. Such techniques include acceleration ofgenetic material coated onto microparticles directly into cells (U.S.Pat. No. 4,945,050 and U.S. Pat. No. 5,141,131). Plants may betransformed using Agrobacterium technology, see U.S. Pat. No. 5,177,010,U.S. Pat. No. 5,104,310, European Patent Application No. 0131624B1,European Patent Application No. 120516, European Patent Application No.159418B1, European Patent Application No. 176112, U.S. Pat. No.5,149,645, U.S. Pat. No. 5,469,976, U.S. Pat. No. 5,464,763, U.S. Pat.No. 4,940,838, U.S. Pat. No. 4,693,976, European Patent Application No.116718, European Patent Application No. 290799, European PatentApplication No. 320500, European Patent Application No. 604662, EuropeanPatent Application No. 627752, European Patent Application No. 0267159,European Patent Application No. 0292435, U.S. Pat. No. 5,231,019, U.S.Pat. No. 5,463,174, U.S. Pat. No. 4,762,785, U.S. Pat. No. 5,004,863,and U.S. Pat. No. 5,159,135. Other transformation technology includesWHISKERS™ technology, see U.S. Pat. No. 5,302,523 and U.S. Pat. No.5,464,765. Electroporation technology has also been used to transformplants, see WO 87/06614, U.S. Pat. No. 5,472,869, U.S. Pat. No.5,384,253, WO 9209696, and WO 9321335. All of these transformationpatents and publications are incorporated by reference. In addition tonumerous technologies for transforming plants, the type of tissue whichis contacted with the foreign genes may vary as well. Such tissue wouldinclude but would not be limited to embryogenic tissue, callus tissuetypes I and II, hypocotyl, meristem, and the like. Almost all planttissues may be transformed during dedifferentiation using appropriatetechniques within the skill of an artisan.

Genes encoding any of the subject toxins can be inserted into plantcells using a variety of techniques which are well known in the art asdisclosed above. For example, a large number of cloning vectorscomprising a marker that permits selection of the transformed microbialcells and a replication system functional in Escherichia coli areavailable for preparation and modification of foreign genes forinsertion into higher plants. Such manipulations may include, forexample, the insertion of mutations, truncations, additions, orsubstitutions as desired for the intended use. The vectors comprise, forexample, pBR322, pUC series, M13mp series, pACYC184, etc. Accordingly,the sequence encoding the Cry protein or variants can be inserted intothe vector at a suitable restriction site. The resulting plasmid is usedfor transformation of cells of E. coli, the cells of which arecultivated in a suitable nutrient medium, then harvested and lysed sothat workable quantities of the plasmid are recovered. Sequenceanalysis, restriction fragment analysis, electrophoresis, and otherbiochemical-molecular biological methods are generally carried out asmethods of analysis. After each manipulation, the DNA sequence used canbe cleaved and joined to the next DNA sequence. Each manipulated DNAsequence can be cloned in the same or other plasmids.

The use of T-DNA-containing vectors for the transformation of plantcells has been intensively researched and sufficiently described in EP120516; Lee and Gelvin (2008), Fraley et al. (1986), and An et al.(1985), and is well established in the field.

Once the inserted DNA has been integrated into the plant genome, it isrelatively stable throughout subsequent generations. The vector used totransform the plant cell normally contains a selectable marker geneencoding a protein that confers on the transformed plant cellsresistance to a herbicide or an antibiotic, such as bialaphos,kanamycin, G418, bleomycin, or hygromycin, inter alia. The individuallyemployed selectable marker gene should accordingly permit the selectionof transformed cells while the growth of cells that do not contain theinserted DNA is suppressed by the selective compound.

A large number of techniques are available for inserting DNA into a hostplant cell. Those techniques include transformation with T-DNA deliveredby Agrobacterium tumefaciens or Agrobacterium rhizogenes as thetransformation agent. Additionally, fusion of plant protoplasts withliposomes containing the DNA to be delivered, direct injection of theDNA, biolistics transformation (microparticle bombardment), orelectroporation, as well as other possible methods, may be employed.

In a preferred embodiment of the subject invention, plants will betransformed with genes wherein the codon usage of the protein codingregion has been optimized for plants. See, for example, U.S. Pat. No.5,380,831, which is hereby incorporated by reference. Also,advantageously, plants encoding a truncated toxin will be used. Thetruncated toxin typically will encode about 55% to about 80% of the fulllength toxin. Methods for creating synthetic B.t. genes for use inplants are known in the art (Stewart, 2007).

Regardless of transformation technique, the gene is preferablyincorporated into a gene transfer vector adapted to express the B.tinsecticidal toxin genes and variants in the plant cell by including inthe vector a plant promoter. In addition to plant promoters, promotersfrom a variety of sources can be used efficiently in plant cells toexpress foreign genes. For example, one may use promoters of bacterialorigin, such as the octopine synthase promoter, the nopaline synthasepromoter, and the mannopine synthase promoter.Non-Bacillus-thuringiensis promoters can be used in some preferredembodiments. Promoters of plant virus origin may be used, for example,the 35S and 19S promoters of Cauliflower Mosaic Virus, a promoter fromCassava Vein Mosaic Virus, and the like. Plant promoters include, butare not limited to, ribulose-1,6-bisphosphate (RUBP) carboxylase smallsubunit (ssu), beta-conglycinin promoter, phaseolin promoter, ADH(alcohol dehydrogenase) promoter, heat-shock promoters, ADF (actindepolymerization factor) promoter, ubiquitin promoter, actin promoter,and tissue specific promoters. Promoters may also contain certainenhancer sequence elements that may improve the transcriptionefficiency. Typical enhancers include but are not limited to ADH1-intron1 and ADH1-intron 6. Constitutive promoters may be used. Constitutivepromoters direct continuous gene expression in nearly all cells typesand at nearly all times (e.g., actin, ubiquitin, CaMV 35S). Tissuespecific promoters are responsible for gene expression in specific cellor tissue types, such as the leaves or seeds (e.g. zein, oleosin, napin,ACP (Acyl Carrier Protein) promoters), and these promoters may also beused. Promoters may also be used that are active during a certain stageof the plants' development as well as active in specific plant tissuesand organs. Examples of such promoters include but are not limited topromoters that are root specific, pollen-specific, embryo specific, cornsilk specific, cotton fiber specific, seed endosperm specific, phloemspecific, and the like.

Under certain circumstances it may be desirable to use an induciblepromoter. An inducible promoter is responsible for expression of genesin response to a specific signal, such as: physical stimulus (e.g. heatshock genes); light (e.g. RUBP carboxylase); hormone (e.g.glucocorticoid); antibiotic (e.g. tetracycline); metabolites; and stress(e.g. drought). Other desirable transcription and translation elementsthat function in plants may be used, such as 5′ untranslated leadersequences, RNA transcription termination sequences and poly-adenylateaddition signal sequences. Numerous plant-specific gene transfer vectorsare known to the art.

Transgenic crops containing insect resistance (IR) traits are prevalentin corn and cotton plants throughout North America, and usage of thesetraits is expanding globally. Commercial transgenic crops combining IRand herbicide tolerance (HT) traits have been developed by multiple seedcompanies. These include combinations of IR traits conferred by B.t.insecticidal proteins and HT traits such as tolerance to AcetolactateSynthase (ALS) inhibitors such as sulfonylureas, imidazolinones,triazolopyrimidine, sulfonanilides, and the like, Glutamine Synthetase(GS) inhibitors such as bialaphos, glufosinate, and the like,4-HydroxyPhenylPyruvate Dioxygenase (HPPD) inhibitors such asmesotrione, isoxaflutole, and the like,5-EnolPyruvylShikimate-3-Phosphate Synthase (EPSPS) inhibitors such asglyphosate and the like, and Acetyl-Coenzyme A Carboxylase (ACCase)inhibitors such as haloxyfop, quizalofop, diclofop, and the like. Otherexamples are known in which transgenically provided proteins provideplant tolerance to herbicide chemical classes such as phenoxy acidsherbicides and pyridyloxyacetates auxin herbicides (see WO 2007/053482A2), or phenoxy acids herbicides and aryloxyphenoxypropionatesherbicides (see WO 2005107437 A2, A3). The ability to control multiplepest problems through IR traits is a valuable commercial productconcept, and the convenience of this product concept is enhanced ifinsect control traits and weed control traits are combined in the sameplant. Further, improved value may be obtained via single plantcombinations of IR traits conferred by a B.t. insecticidal protein suchas that of the subject invention, with one or more additional HT traitssuch as those mentioned above, plus one or more additional input traits(e.g. other insect resistance conferred by B.t.-derived or otherinsecticidal proteins, insect resistance conferred by mechanisms such asRNAi and the like, nematode resistance, disease resistance, stresstolerance, improved nitrogen utilization, and the like), or outputtraits (e.g. high oils content, healthy oil composition, nutritionalimprovement, and the like). Such combinations may be obtained eitherthrough conventional breeding (breeding stack) or jointly as a noveltransformation event involving the simultaneous introduction of multiplegenes (molecular stack). Benefits include the ability to manage insectpests and improved weed control in a crop plant that provides secondarybenefits to the producer and/or the consumer. Thus, the subjectinvention can be used in combination with other traits to provide acomplete agronomic package of improved crop quality with the ability toflexibly and cost effectively control any number of agronomic issues.

The transformed cells grow inside the plants in the usual manner. Theycan form germ cells and transmit the transformed trait(s) to progenyplants.

Such plants can be grown in the normal manner and crossed with plantsthat have the same transformed hereditary factors or other hereditaryfactors. The resulting hybrid individuals have the correspondingphenotypic properties.

In a preferred embodiment of the subject invention, plants will betransformed with genes wherein the codon usage has been optimized forplants. See, for example, U.S. Pat. No. 5,380,831. In addition, methodsfor creating synthetic Bt genes for use in plants are known in the art(Stewart and Burgin, 2007). One non-limiting example of a preferredtransformed plant is a fertile maize plant comprising a plantexpressible gene encoding a Cry6Aa protein, and further comprising asecond set of plant expressible genes encoding Cry34Ab/35Ab proteins.

Transfer (or introgression) of the Cry6Aa- and Cry34Ab/35Ab-determinedtrait(s) into inbred maize lines can be achieved by recurrent selectionbreeding, for example by backcrossing. In this case, a desired recurrentparent is first crossed to a donor inbred (the non-recurrent parent)that carries the appropriate gene(s) for the Cry-determined traits. Theprogeny of this cross is then mated back to the recurrent parentfollowed by selection in the resultant progeny for the desired trait(s)to be transferred from the non-recurrent parent. After three, preferablyfour, more preferably five or more generations of backcrosses with therecurrent parent with selection for the desired trait(s), the progenywill be heterozygous for loci controlling the trait(s) beingtransferred, but will be like the recurrent parent for most or almostall other genes (see, for example, Poehlman & Sleper (1995) BreedingField Crops, 4th Ed., 172-175; Fehr (1987) Principles of CultivarDevelopment, Vol. 1: Theory and Technique, 360-376).

Insect Resistance Management (IRM) Strategies. Roush et al., forexample, outlines two-toxin strategies, also called “pyramiding” or“stacking,” for management of insecticidal transgenic crops. (The RoyalSociety. Phil. Trans. R. Soc. Lond. B. (1998) 353, 1777-1786).

On their website, the United States Environmental Protection Agency(epa.gov/oppbppd1/biopesticides/pips/bt_corn_refuge_(—)2006.htm)publishes the following requirements for providing non-transgenic (i.e.,non-B.t.) refuges (a block of non-Bt crops/corn) for use with transgeniccrops producing a single Bt protein active against target pests.

-   -   “The specific structured requirements for corn borer-protected        Bt (Cry1Ab or Cry1F) corn products are as follows:    -   Structured refuges: 20% non-Lepidopteran Bt corn refuge in Corn        Belt;        -   50% non-Lepidopteran Bt refuge in Cotton Belt    -   Blocks        -   Internal (i.e., within the Bt field)        -   External (i.e., separate fields within ½ mile (¼ mile if            possible) of the Bt field to maximize random mating)    -   In-field Strips        -   Strips must be at least 4 rows wide (preferably 6 rows) to            reduce the effects of larval movement”

In addition, the National Corn Growers Association, on their website:

-   -   (ncga.com/insect-resistance-management-fact-sheet-bt-corn)        also provides similar guidance regarding the refuge        requirements. For example:    -   “Requirements of the Corn Borer IRM:    -   Plant at least 20% of your corn acres to refuge hybrids    -   In cotton producing regions, refuge must be 50%    -   Must be planted within ½ mile of the refuge hybrids    -   Refuge can be planted as strips within the Bt field; the refuge        strips must be at least 4 rows wide    -   Refuge may be treated with conventional pesticides only if        economic thresholds are reached for target insect    -   Bt-based sprayable insecticides cannot be used on the refuge        corn    -   Appropriate refuge must be planted on every farm with Bt corn”

As stated by Roush et al. (on pages 1780 and 1784 right column, forexample), stacking or pyramiding of two different proteins eacheffective against the target pests and with little or nocross-resistance can allow for use of a smaller refuge. Roush suggeststhat for a successful stack, a refuge size of less than 10% refuge, canprovide comparable resistance management to about 50% refuge for asingle (non-pyramided) trait. For currently available pyramided Bt cornproducts, the U.S. Environmental Protection Agency requiressignificantly less (generally 5%) structured refuge of non-Bt corn beplanted than for single trait products (generally 20%).

There are various ways of providing the IRM effects of a refuge,including various geometric planting patterns in the fields (asmentioned above) and in-bag seed mixtures, as discussed further by Roushet al. (supra), and U.S. Pat. No. 6,551,962.

The above percentages, or similar refuge ratios, can be used for thesubject double or triple stacks or pyramids.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety to the extent they are not inconsistent with theexplicit teachings of this specification.

Following are examples that illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted. All temperatures are in degrees Celsius.

Unless specifically indicated or implied, the terms “a”, “an”, and “the”signify “at least one” as used herein.

EXAMPLES Example 1 Construction of Expression Plasmids EncodingCry34Ab1, Cry35Ab1, and Cry6Aa1 Full-Length Toxins

Standard cloning methods were used in the construction of Pseudomonasfluorescens (Pf) expression plasmids engineered to produce a full-lengthCry34Ab1, Cry35Ab1, and Cry6Aa1 Cry proteins, respectively. Restrictionendonucleases from New England BioLabs (NEB; Ipswich, Mass.) were usedfor DNA digestion and T4 DNA Ligase from Invitrogen was used for DNAligation. Plasmid preparations were performed using the Plasmid Mini kit(Qiagen, Valencia, Calif.), following the instructions of the supplier.DNA fragments were purified using the Millipore Ultrafree®-DA cartridge(Billerica, Mass.) after agarose Tris-acetate gel electrophoresis. Thebasic cloning strategy entailed subcloning the coding sequences (CDS) ofa full-length of these Cry proteins into pMYC1803 for Cry34Ab1 andCry35Ab1, and into pDOW1169 for Cry6Aa1 at SpeI and XhoI (or SalI thatis compatible with XhoI) restriction sites, respectively, whereby theywere placed under the expression control of the Ptac promoter and therrnBT1T2 or rrnBT2 terminator from plasmid pKK223-3 (PL Pharmacia,Milwaukee, Wis.), respectively. pMYC1803 is a medium copy number plasmidwith the RSF1010 origin of replication, a tetracycline resistance gene,and a ribosome binding site preceding the restriction enzyme recognitionsites into which DNA fragments containing protein coding regions may beintroduced (US Patent Application No. 20080193974).

The expression plasmids for both Cry34Ab1 and Cry35Ab1 were transformedby electroporation into a P. fluorescens strain MB214, recovered inSOC-Soy hydrolysate medium, and plated on Lysogeny broth (LB) mediumcontaining 20 μg/ml tetracycline. The expression vector pDOW1169 issimilar to pMYC1803 but pDOW1169 carries a gene pyrF encoding uracil,which was used as a marker for screening for transformants when a P.fluorescens uracil auxotrophic strain (such as DPf10) was used fortransformation on a plate of M9 minimal medium that lacked of uracil(Schneider et al. 2005). Details of the microbiological manipulationsare available from US Patent Application No. 20060008877, US PatentApplication No. 20080193974, and US Patent Application No. 20080058262,incorporated herein by reference. Colonies were further screened byrestriction digestion of miniprep plasmid DNA. Plasmid DNA of selectedclones containing inserts was sequenced by contract with a commercialsequencing vendor such as eurofins MWG Operon (Huntsville, Ala.).Sequence data were assembled and analyzed using the Sequencher™ software(Gene Codes Corp., Ann Arbor, Mich.).

Example 2 Growth and Expression

Growth and expression analysis in shake flasks production of Cry34Ab1,Cry35Ab1, and Cry6Aa1 toxins for characterization including Bt receptorbinding and insect bioassay was accomplished by shake-flask-grown P.fluorescens strains harboring expression constructs (e.g. clone pMYC2593for Cry34Ab1, pMYC3122 for Cry35Ab1, and pDAB102018 for Cry6Aa1). Seedcultures for Cry34Ab1 and Cry35Ab1 grown in P. fluorescens mediumovernight supplemented with 20 μg/ml tetracycline were used to inoculate200 mL of the same medium with 20 μg/ml tetracycline. However, the seedculture for Cry6Aa1 was grown in M9 minimal broth overnight and was usedto inoculate 200 mL of the P. fluorescens medium without antibiotic.Expressions of Cry34Ab1, Cry35Ab1, and Cry6Aa1 toxins via the Ptacpromoter were induced by addition ofisopropyl-β-D-1-thiogalactopyranoside (IPTG) after an initial incubationof 24 hours at 28-30° C. with shaking at 300 rpm. Cultures were sampledat the time of induction and at various times post-induction. Celldensity was measured by optical density at 600 nm (OD₆₀₀).

Example 3 Cell Fractionation and SDS-PAGE Analysis of Shake FlaskSamples

At each sampling time, the cell density of the samples was adjusted toOD₆₀₀=20 and 1-mL aliquots are centrifuged at 14,000×g for five minutes.The cell pellets were frozen at −80° C. Soluble and insoluble fractionsfrom frozen cell pellet samples were generated using EasyLyse™ BacterialProtein Extraction Solution (EPICENTRE® Biotechnologies, Madison, Wis.).Each cell pellet was resuspended in 1 mL EasyLyse™ solution and furtherdiluted 1:4 in lysis buffer and incubated with shaking at roomtemperature for 30 minutes. The lysate was centrifuged at 14,000 rpm for20 minutes at 4° C. and the supernatant was recovered as the solublefraction. The pellet (insoluble fraction) was then resuspended in anequal volume of phosphate buffered saline (PBS; 11.9 mM Na₂HPO₄, 137 mMNaCl, 2.7 mM KCl, pH7.4). Samples were mixed at 3:1 with 4× Laemmlisample buffer containing β-mercaptoethanol and boiled for 5 minutesprior to loading onto NuPAGE Novex 4-20% Bis-Tris gels (Invitrogen,Carlsbad, Calif.). Electrophoresis was performed in the recommendedNuPAGE MOPS buffer. Gels were stained with the SimplyBlue™ Safe Stainaccording to the manufacturer's (Invitrogen) protocol and imaged usingthe Typhoon imaging system (GE Healthcare Life Sciences, Pittsburgh,Pa.).

Example 4 Inclusion Body Preparation

Cry protein inclusion body (IB) preparations were performed from P.fluorescens fermentations that produced insoluble B.t. insecticidalprotein, as demonstrated by SDS-PAGE and MALDI-MS (Matrix Assisted LaserDesorption/Ionization Mass Spectrometry). P. fluorescens cell pelletscreated from 48 hours post induction were thawed in a 37° C. water bath.The cells were resuspended to 25% w/v in lysis buffer (50 mM Tris, pH7.5, 200 mM NaCl, 20 mM EDTA disodium salt (Ethylenediaminetetraaceticacid), 1% Triton X-100, and 5 mM Dithiothreitol (DTT); 5 mL/L ofbacterial protease inhibitor cocktail (P8465 Sigma-Aldrich, St. Louis,Mo.) was added just prior to use only for Cry34Ab1 and Cry35Ab1. Thecells were suspended using a homogenizer at lowest setting (TissueTearor, BioSpec Products, Inc., Bartlesville, Okla.). Twenty five mg oflysozyme (Sigma L7651, from chicken egg white) was added to the cellsuspension by mixing with a metal spatula, and the suspension wasincubated at room temperature for one hour. The suspension was cooled onice for 15 minutes, then sonicated using a Branson Sonifier 250 (two1-minute sessions, at 50% duty cycle, 30% output). Cell lysis waschecked by microscopy. An additional 25 mg of lysozyme was added ifnecessary, and the incubation and sonication were repeated. When celllysis was confirmed via microscopy, the lysate was centrifuged at11,500×g for 25 minutes (4° C.) to form the IB pellet, and thesupernatant was discarded. The IB pellet was resuspended with 100 mLlysis buffer, homogenized with the hand-held mixer and centrifuged asabove. The IB pellet was repeatedly washed by resuspension (in 50 mLlysis buffer), homogenization, sonication, and centrifugation until thesupernatant became colorless and the IB pellet became firm and off-whitein color. For the final wash, the IB pellet was resuspended insterile-filtered (0.22 μm) distilled water containing 2 mM EDTA, andcentrifuged. The final pellet was resuspended in sterile-filtereddistilled water containing 2 mM EDTA, and stored in 1 mL aliquots at−80° C.

Example 5 SDS-PAGE Analysis and Quantification

SDS-PAGE analysis and quantification of protein in IB preparations weredone by thawing a 1 mL aliquot of IB pellet and diluting 1:20 withsterile-filtered distilled water. The diluted sample was then boiledwith 4× reducing sample buffer [250 mM Tris, pH6.8, 40% glycerol (v/v),0.4% Bromophenol Blue (w/v), 8% SDS (w/v) and 8% β-Mercapto-ethanol(v/v)] and loaded onto a Novex® 4-20% Tris-Glycine, 12+2 well gel(Invitrogen) run with 1× Tris/Glycine/SDS buffer (Invitrogen). The gelwas run for approximately 60 min at 200 volts then stained and distainedby following the SimplyBlue™ Safe Stain (Invitrogen) procedures.Quantification of target bands was done by comparing densitometricvalues for the bands against Bovine Serum Albumin (BSA) samples run onthe same gel to generate a standard curve using the Bio-Rad Quantity Onesoftware.

Example 6 Solubilization of Inclusion Bodies

Ten mL of inclusion body suspensions from P. fluorescens clones MR1253,MR1636, and DPf13032 (containing 50-70 mg/mL of Cry34Ab1, Cry35Ab1, andCry6Aa1 proteins respectively) were centrifuged at the highest settingof an Eppendorf model 5415C microfuge (approximately 14,000×g) to pelletthe inclusions. The storage buffer supernatant was removed and replacedwith 25 mL of 100 mM sodium acetate buffer, pH 3.0, for both Cry34Ab1and Cry35Ab1, and 50 mM CAPS [3-(cyclohexamino)1-propanesulfonic acid]buffer, pH10.5, for Cry6Aa1, in a 50 mL conical tube, respectively.Inclusions were resuspended using a pipette and vortexed to mixthoroughly. The tubes were placed on a gently rocking platform at 4° C.overnight to extract full-length Cry34Ab1, Cry35Ab1, and Cry6Aa1proteins. The extracts were centrifuged at 30,000×g for 30 min at 4° C.,and saved the resulting supernatants containing solubilized full-lengthCry proteins.

Example 7 Truncation of Full-Length Protoxin

Full-length Cry35Ab1 was truncated or digested with chymotrypsin to getits chymotrypsin core that is an active form of the Cry protein.Specifically, the solubilized full-length Cry35Ab1 was incubated withchymotrypsin (bovine pancreas) (Sigma, St. MO) at (50:1=Cryprotein:enzyme, w/w) in the 100 mM sodium acetate buffer, pH 3.0, at 4°C. with gentle shaking for 2-3 days, Complete activation or truncationwas confirmed by SDS-PAGE analysis. The molecular mass of thefull-length Cry35Ab1 was ≈44 kDa, and the chymotrypsin core was ≈40 kDa.The amino acid sequences of the full-length and chymotrypsin core areprovided as SEQ ID NO:1 and SEQ ID NO:2. Either chymotrypsin or trypsincore is not available for Cry34Ab1, and also full-length Cry6Aa1 issignificantly more active to target insect corn rootworm than either itschymotrypsin or trypsin core. Thus, the full-length Cry34Ab1 and Cry6Aa1were used for binding assays. The amino acid sequences of thefull-length Cry34Ab1 and Cry6Aa1 are provided as SEQ ID NO:3 and SEQ IDNO:4.

Example 8 Purification of Cry Toxins

The chymotrypsinized Cry35Ab1 and full-length Cry6Aa1 were furtherpurified using an ion-exchange chromatography system. Specifically, theCry35Ab1 digestion reaction was centrifuged at 30,000×g for 30 min at 4°C. and followed by going through a 0.22 μm filter to remove lipids andall other particles, and the resulting solution was concentrated 5-foldusing an Amicon Ultra-15 regenerated cellulose centrifugal filter device(10,000 Molecular Weight Cutoff; Millipore). The sample buffers werethen changed to 20 mM sodium acetate buffer, pH 3.5, for both Cry34Ab1and Cry35Ab1, using disposable PD-10 columns (GE Healthcare, Piscataway,N.J.) or dialysis. They were further purified using ATKA Explorer liquidchromatography system (Amersham Biosciences). For Cry35Ab1, the buffer Awas 20 mM sodium acetate buffer, pH 3.5, and the buffer B was the bufferA+1 M NaCl, pH 3.5, while for Cry6Aa1 the buffer A was 50 mM CAPS, pH10.5 and the buffer B was 50 mM CAPS, pH 10.5, 1 M NaCl. A HiTrap SP (5ml) column (GE) was used for truncated Cry35Ab1. After the column wasfully equilibrated using the buffer A, the Cry35Ab1 solution wasinjected into the column at a flow rate of 5 ml/min. Elution wasperformed using gradient 0-100% of the buffer B at 5 ml/min with 1ml/fraction.

For full-length Cry6Aa1, a Capto Q, 5 ml (5 ml) column (GE) was used andthe all other procedures were similar to those for Cry35Ab1. AfterSDS-PAGE analysis of the selected fractions to further select fractionscontaining the best quality target protein, pooled those fractions. Thebuffer was changed for the purified Cry35Ab1 chymotrypsin core with 20mM Bist-Tris, pH 6.0, as described above. For the purified Cry6Aa1, thesalt was removed through dialysis against 10 mM CAPS, pH 10.5. Thesamples were saved at 4° C. for later binding assay after beingquantified using SDS-PAGE and the Typhoon imaging system (GE) analyseswith BSA as a standard. Full-length Cry34Ab1 was already pure afterbeing solubilized in the acidic buffer as judged by SDS-PAGE analysis,and thus without further purification.

Example 9 BBMV Preparations

Brush border membrane vesicle (BBMV) preparations of insect midguts havebeen widely used for Cry toxin receptor binding assays. The BBMVpreparations used in this invention were prepared from isolated midgutsof third instars of the western corn rootworm (Diabrotica virgiferavirgifera LeConte) using the method described by Wolferberger et al.(1987). Leucine aminopeptidase was used as a marker of membrane proteinsin the preparation and Leucine aminopeptidase activities of crudehomogenate and BBMV preparation were determined as previously described(Li et al. 2004a). Protein concentration of the BBMV preparation wasmeasured using the Bradford method (1976).

Example 10 ¹²⁵I Labeling

Purified full-length Cry34Ab1, chymotrypsinized Cry35Ab1, andfull-length Cry6Aa1 were labeled using ¹²⁵I for saturation andcompetition binding assays. To ensure the radio-labeling does notabolish the biological activity of the Cry toxins, cold iodination wasconducted using NaI by following the instructions of Pierce® IodinationBeads (Pierce Biotechnology, Thermo Scientific, Rockford Ill.). Bioassayresults indicated that both iodinated Cry35Ab1 chymotrypsin core andfull-length Cry6Aa1 remained active against the larvae of the westerncorn rootworm, but iodination inactivated Cry34Ab1. As expected,¹²⁵I-Cry34Ab1 did not specifically bind to the insect BBMV, and thusCry34Ab1 requires another labeling method to assess membrane receptorbinding. ¹²⁵I-Cry35Ab1 and ¹²⁵I-Cry6Aa1 were obtained with Pierce®Iodination Beads (Pierce) and Na¹²⁵I. Zeba™ Desalt Spin Columns (Pierce)were used to remove unincorporated or free Na¹²⁵I from the iodinatedprotein. The specific radio-activities of the iodinated Cry proteinsranged from 1-5 uCi/ug. Multiple batches of labeling and binding assayswere conducted.

Example 11 Saturation Binding Assays

Saturation binding assays were performed using ¹²⁵I-labeled Cry toxinsas described previously (Li et al. 2004b). To determine specific bindingand estimate the binding affinity (disassociation constant, Kd) andbinding site concentration (the amount of toxin specifically bound to agiven amount of BBMV, Bmax) of Cry35Ab1 and Cry6Aa1 to the insect BBMV,a series of increasing concentrations of either ¹²⁵I-Cry35Ab1 or¹²⁵I-Cry6Aa1 were incubated with a given concentration (0.1 mg/ml) ofthe insect BBMV, respectively, in 150 ul of 20 mM Bis-Tris, pH 6.0, 150mM KCl, supplemented with 0.1% BSA at room temperature for 60 min withgentle shaking. Toxin bound to BBMV was separated from free toxins inthe suspension by centrifugation at 20,000×g at room temperature for 8min. The pellet was washed twice with 900 ul of ice-cold the same buffercontaining 0.1% BSA. The radio-activity remaining in the pellet wasmeasured with a COBRAII Auto-Gamma counter (Packard, a Canberra company)and considered total binding.

Another series of binding reactions were setup at side by side, and a500-1,000-fold excess of unlabeled corresponding toxin was included ineach of the binding reactions to fully occupy all specific binding siteson the BBMV, which was used to determine non-specific binding. Specificbinding was estimated by subtracting the non-specific binding from thetotal binding. The Kd and Bmax values of these toxins were estimatedusing the toxin molecule number (pmole) specifically bound to permicrogram BBMV protein against the concentrations of the labeled toxinused by running GraphPad Prism 5.01 (GraphPad Software, San Diego,Calif.). The charts were made using either Microsoft Excel or GraphPadPrism program. The experiments were replicated at least three times.These binding experiments demonstrated that both ¹²⁵I-Cry35Ab1 and¹²⁵I-Cry6Aa1 were able to specifically bind to the BBMV (FIGS. 1A and1B). ¹²⁵I-Cry35Ab1 and ¹²⁵I-Cry6Aa1 had a binding affinityKd=11.66±11.35, 7.99±4.89 (nM), respectively, and a binding siteconcentration Bmax=5.19±3.02, 2.71±0.90 (pmole/ug BBMV), respectively.

Example 12 Competition Binding Assays

Competition binding assays were further conducted to determine ifCry35Ab1 and Cry6Aa1 share a same set of receptors. For Cry35Ab1homologous competition binding assays, increasing amounts (0-5,000 nM)of unlabeled Cry35Ab1 were first mixed with 5 nM labeled Cry35Ab1, andthen incubated with a given concentration (0.1 mg/ml) of BBMV at roomtemperature for 60 min, respectively. The percentages of bound¹²⁵I-Cry35Ab1 with BBMV were determined for each of the reactions ascompared to the initial specific binding at absence of unlabeledcompetitor. Heterologous competition binding assay between ¹²⁵I-Cry35Ab1and unlabeled Cry6Aa1 was performed to identify if they share a same setof receptor(s). This was achieved by increasing the amount of unlabeledCry6Aa1 as a competitor included in the reactions to compete for theputative receptor(s) on the BBMV with the labeled Cry35Ab1. Theexperiment was replicated at least three times.

The experimental results demonstrated that Cry35Ab1 was able to displaceitself over 50% when the molar concentration increased to approximately100 nM (20 folds excess compared to 5 nM ¹²⁵I-Cry35Ab1). The remainingabout 50% was considered nonspecific binding that was not able to bedisplaced based on the saturation binding result as described above.This suggests that the specific binding was completely displaced by20-fold excess unlabeled Cry35Ab1 (FIG. 2). However, Cry6Aa1 was notable to displace ¹²⁵I-Cry35Ab1. These data indicate that Cry35Ab1 doesnot share a receptor with Cry6Aa1. Whether or not Cry34Ab1 and Cry6Aa1share a receptor remains to test using unlabeled Cry34Ab1 to compete forthe binding with radio-labeled Cry6Aa or unlabeled Cry6Aa1 to competefor the binding with labeled Cry34Ab1 with another labeling method.

REFERENCES

-   Bradford, M. M. 1976. A rapid and sensitive method for the    quantitation of microgram quantities of protein utilizing the    principle of protein-dye binding, Anal. Biochem. 72, 248-254.-   Li, H., Oppert, B., Higgins, R. A., Huang, F., Zhu, K. Y.,    Buschman, L. L., 2004a. Comparative analysis of proteinase    activities of Bacillus thuringiensis-resistant and -susceptible    Ostrinia nubilalis (Lepidoptera: Crambidae). Insect Biochem. Mol.    Biol. 34, 753-762.

Li, H., Oppert, B., Gonzalez-Cabrera, J., Ferré, J., Higgins, R. A.,Buschman, L. L. and Zhu, K. Y. and Huang, F. 2004b. Binding analysis ofCry1Ab and Cry1Ac with membrane vesicles from Bacillusthuringiensis-resistant and -susceptible Ostrinia nubilalis(Lepidoptera: Crambidae). Biochem. Biophys. Res. Commun. 323, 52-57.

-   Schneider, J. C. Jenings A F, Mun D M, McGovern P M, Chew L C. 2005.    Auxotrophic markers pyrF and proC can replace antibiotic markers on    protein production plasmids in high-cell-density Pseudomonas    fluorescens fermentation. Biotechnology Progress 21, 343-348.-   Wolfersberger, M. G., Luthy, P., Maurer, A., Parenti, P., Sacchi,    F., Giordana, B., Hanozet, G. M., 1987. Preparation and partial    characterization of amino acid transporting brush border membrane    vesicles from the larval midgut of the cabbage butterfly (Pieris    brassicae). Comp. Biochem. Physiol. 86A, 301-308.-   US Patent Application No. 20080193974.2008. BACTERIAL LEADER    SEQUENCES FOR INCREASED EXPRESSION-   US Patent Application No. 20060008877, 2006. Expression systems with    sec-system secretion.-   US Patent Application No. 20080058262, 2008. rPA optimization.

1. A transgenic plant that produces a Cry34 protein, a Cry35 protein,and a Cry6A insecticidal protein.
 2. The transgenic plant of claim 1,said plant further produces a fourth insecticidal protein selected fromthe group consisting of Cry3B and Cry3A.
 3. Seed of a plant according toclaim 1, wherein said seed comprises DNA encoding said proteins.
 4. Afield of plants comprising a plurality of plants according to claim 1.5. The field of plants of claim 4, said field further comprising non-Btrefuge plants, wherein said refuge plants comprise less than 40% of allcrop plants in said field.
 6. The field of plants of claim 5, whereinsaid refuge plants comprise less than 30% of all crop plants in saidfield.
 7. The field of plants of claim 5, wherein said refuge plantscomprise less than 20% of all crop plants in said field.
 8. The field ofplants of claim 5, wherein said refuge plants comprise less than 10% ofall crop plants in said field.
 9. The field of plants of claim 5,wherein said refuge plants comprise less than 5% of all crop plants insaid field.
 10. The field of plants of claim 4, wherein said field lacksrefuge plants.
 11. The field of plants of claim 5, wherein said refugeplants are in blocks or strips.
 12. A mixture of seeds comprising refugeseeds from non-Bt refuge plants, and a plurality of seeds of claim 3,wherein said refuge seeds comprise less than 40% of all the seeds in themixture.
 13. The mixture of seeds of claim 12, wherein said refuge seedscomprise less than 30% of all the seeds in the mixture.
 14. The mixtureof seeds of claim 12, wherein said refuge seeds comprise less than 20%of all the seeds in the mixture.
 15. The mixture of seeds of claim 12,wherein said refuge seeds comprise less than 10% of all the seeds in themixture.
 16. The mixture of seeds of claim 12, wherein said refuge seedscomprise less than 5% of all the seeds in the mixture.
 17. A seed bag orcontainer comprising a plurality of seeds of claim 3, said bag orcontainer having zero refuge seed.
 18. A method of managing developmentof resistance to a Cry protein by an insect, said method comprisingplanting seeds to produce a field of plants of claim
 4. 19. A field ofclaim 4, wherein said plants occupy more than 10 acres.
 20. A plant ofclaim 1, wherein said plant is a maize plant.
 21. A plant cell of aplant of claim 1, wherein said Cry35 protein is at least 95% identicalwith a sequence selected from the group consisting of SEQ ID NO:1 andSEQ ID NO:2, said Cry6A insecticidal protein is at least 95% identicalwith SEQ ID NO:2, and said Cry34Ab protein is at least 95% identicalwith SEQ ID NO:3.
 22. A plant of claim 1, wherein said Cry35 proteincomprises a sequence selected from the group consisting of SEQ ID NO:1and SEQ ID NO:2, said Cry6A insecticidal protein comprises SEQ ID NO:2,and said Cry34 protein comprises SEQ ID NO:3.
 23. A method of producingthe plant cell of claim
 21. 24. A method of controlling a rootworminsect by contacting said insect with a Cry34 protein, a Cry35 protein,and a Cry6A insecticidal protein.
 25. The plant of claim 1 wherein saidCry34 protein is a Cry34A protein, said Cry35 protein is a Cry35Aprotein, and said Cry6A protein is a Cry6Aa protein.
 26. The plant ofclaim 1 wherein said Cry34 protein is a Cry34Aa protein and said Cry35protein is a Cry35Aa protein.
 27. The plant of claim 2 wherein saidCry3A protein is a Cry3Aa protein and said Cry3B protein is a Cry3Baprotein.
 28. The method of claim 24 wherein said Cry34 protein is aCry34A protein, said Cry35 protein is a Cry35A protein, and said Cry6Aprotein is a Cry6Aa protein.
 29. The method of claim 24 wherein saidCry34 protein is a Cry34Aa protein and said Cry35 protein is a Cry35Aaprotein.